A Combined Optogenetic-Knockdown Strategy Reveals a Major Role of Tomosyn in Mossy Fiber Synaptic Plasticity.

Neurotransmitter release probability (P(r)) largely determines the dynamic properties of synapses. While much is known about the role of presynaptic proteins in transmitter release, their specific contribution to synaptic plasticity is unclear. One such protein, tomosyn, is believed to reduce P(r) by interfering with the SNARE complex formation. Tomosyn is enriched at hippocampal mossy fiber-to-CA3 pyramidal cell synapses (MF-CA3), which characteristically exhibit low P(r), strong synaptic facilitation, and pre-synaptic protein kinase A (PKA)-dependent long-term potentiation (LTP). To evaluate tomosyn's role in MF-CA3 function, we used a combined knockdown (KD)-optogenetic strategy whereby presynaptic neurons with reduced tomosyn levels were selectively activated by light. Using this approach in mouse hippocampal slices, we found that facilitation, LTP, and PKA-induced potentiation were significantly impaired at tomosyn-deficient synapses. These findings not only indicate that tomosyn is a key regulator of MF-CA3 plasticity but also highlight the power of a combined KD-optogenetic approach to determine the role of presynaptic proteins.

Compartment-specific modulation of GABAergic synaptic transmission by TRPV1 channels in the dentate gyrus.

The transient receptor potential TRPV1 or vanilloid receptor is a nonselective ligand-gated channel highly expressed in primary sensory neurons where it mediates nociception. TRPV1 is also expressed in the brain where its activation depresses excitatory synaptic transmission. Whether TRPV1 also regulates inhibitory synapses in the brain is unclear. Here, using a combination of pharmacology, electrophysiology, and an in vivo knockdown strategy, we report that TRPV1 activation by capsaicin or by the endocannabinoid anandamide depresses somatic, but not dendritic inhibitory transmission in both rat and mouse dentate gyrus. The effect on somatic inhibition was absent in TRPV1 knock-out mice and was also eliminated by two different TRPV1 shRNAs expressed in dentate granule cells, strongly supporting a functional role for TRPV1 in modulating GABAergic synaptic function. Moreover, TRPV1-mediated depression occurs independently of GABA release, requires postsynaptic Ca(2+) rise and activation of calcineurin, and is likely due to clathrin-dependent internalization of GABA receptors. Altogether, these findings reveal a novel form of compartment-specific regulation whereby TRPV1 channels can modify synaptic function in the brain.

REST-dependent epigenetic remodeling promotes the developmental switch in synaptic NMDA receptors.

NMDA receptors (NMDARs) are critical to synaptogenesis, neural circuitry and higher cognitive functions. A hallmark feature of NMDARs is an early postnatal developmental switch from those containing primarily GluN2B to primarily GluN2A subunits. Although the switch in phenotype has been an area of intense interest for two decades, the mechanisms that trigger it and the link between experience and the switch are unclear. Here we show a new role for the transcriptional repressor REST in the developmental switch of synaptic NMDARs. REST is activated at a critical window of time and acts via epigenetic remodeling to repress Grin2b expression and alter NMDAR properties at rat hippocampal synapses. Knockdown of REST in vivo prevented the decline in GluN2B and developmental switch in NMDARs. Maternal deprivation impaired REST activation and acquisition of the mature NMDAR phenotype. Thus, REST is essential for experience-dependent fine-tuning of genes involved in synaptic plasticity.

SNAP-25 is a target of protein kinase C phosphorylation critical to NMDA receptor trafficking.

Protein kinase C (PKC) enhances NMDA receptor (NMDAR)-mediated currents and promotes NMDAR delivery to the cell surface via SNARE-dependent exocytosis. Although the mechanisms of PKC potentiation are established, the molecular target of PKC is unclear. Here we show that synaptosomal-associated protein of 25 kDa (SNAP-25), a SNARE protein, is functionally relevant to PKC-dependent NMDAR insertion, and identify serine residue-187 as the molecular target of PKC phosphorylation. Constitutively active PKC delivered via the patch pipette potentiated NMDA (but not AMPA) whole-cell currents in hippocampal neurons. Expression of RNAi targeting SNAP-25 or mutant SNAP-25(S187A) and/or acute disruption of the SNARE complex by treatment with BoNT A, BoNT B or SNAP-25 C-terminal blocking peptide abolished NMDAR potentiation. A SNAP-25 peptide and function-blocking antibody suppressed PKC potentiation of NMDA EPSCs at mossy fiber-CA3 synapses. These findings identify SNAP-25 as the target of PKC phosphorylation critical to PKC-dependent incorporation of synaptic NMDARs and document a postsynaptic action of this major SNARE protein relevant to synaptic plasticity.

Regulation of NMDA receptor Ca2+ signalling and synaptic plasticity.

NMDARs (N-methyl-D-aspartate receptors) are critical for synaptic function throughout the CNS (central nervous system). NMDAR-mediated Ca(2+) influx is implicated in neuronal differentiation, neuronal migration, synaptogenesis, structural remodelling, long-lasting forms of synaptic plasticity and higher cognitive functions. NMDAR-mediated Ca(2+) signalling in dendritic spines is not static, but can be remodelled in a cell- and synapse-specific manner by NMDAR subunit composition, protein kinases and neuronal activity during development and in response to sensory experience. Recent evidence indicates that Ca(2+) permeability of neuronal NMDARs, NMDAR-mediated Ca(2+) signalling in spines and induction of NMDAR-dependent LTP (long-term potentiation) at hippocampal Schaffer collateral-CA1 synapses are under control of the cAMP/PKA (protein kinase A) signalling cascade. Thus, by enhancing Ca(2+) influx through NMDARs in spines, PKA can regulate the induction of LTP. An emerging concept is that activity-dependent regulation of NMDAR-mediated Ca(2+) signalling by PKA and by extracellular signals that modulate cAMP or protein phosphatases at synaptic sites provides a dynamic and potentially powerful mechanism for bi-directional regulation of synaptic efficacy and remodelling.

Developmental switch in requirement for PKA RIIbeta in NMDA-receptor-dependent synaptic plasticity at Schaffer collateral to CA1 pyramidal cell synapses.

The cAMP/protein kinase A (PKA) signaling cascade is crucial for synaptic plasticity in a wide variety of species. PKA regulates Ca2+ permeation through NMDA receptors (NMDARs) and induction of NMDAR-dependent synaptic plasticity at the Schaffer collateral to CA1 pyramidal cell synapse. Whereas the role of PKA in induction of NMDAR-dependent LTP at CA1 synapses is established, the identity of PKA isoforms involved in this phenomenon is less clear. Here we report that protein synthesis-independent NMDAR-dependent LTP at the Schaffer collateral-CA1 synapse in the hippocampus is deficient, but NMDAR-dependent LTD is normal, in young (postnatal day 10 (P10)-P14) mice lacking PKA RIIbeta, the PKA regulatory protein that links PKA to NMDARs at synaptic sites. In contrast, in young adult (P21-P28) mice lacking PKA RIIbeta, LTP is normal and LTD is abolished. These findings indicate that distinct PKA isoforms may subserve distinct forms of synaptic plasticity and are consistent with a developmental switch in the signaling cascades required for LTP induction.

Distinct roles for ephrinB3 in the formation and function of hippocampal synapses.

The transmembrane ephrinB ligands and their Eph receptor tyrosine kinases are known to regulate excitatory synaptic functions in the hippocampus. In the CA3-CA1 synapse, ephrinB ligands are localized to the post-synaptic membrane, while their cognate Eph receptors are presumed to be pre-synaptic. Interaction of ephrinB molecules with Eph receptors leads to changes in long-term potentiation (LTP), which has been reported to be mediated by reverse signaling into the post-synaptic membrane. Here, we demonstrate that the cytoplasmic domain of ephrinB3 and hence reverse signaling is not required for ephrinB dependent learning and memory tasks or for LTP of these synapses. Consistent with previous reports, we find that ephrinB3(KO) null mutant mice exhibit a striking reduction in CA3-CA1 LTP that is associated with defective learning and memory tasks. We find the null mutants also show changes in both pre- and post-synaptic proteins including increased levels of synapsin and synaptobrevin and reduced levels of NMDA receptor subunits. These abnormalities are not observed in ephrinB3(lacZ) reverse signaling mutants that specifically delete the ephrinB3 intracellular region, supporting a cytoplasmic domain-independent forward signaling role for ephrinB3 in these processes. We also find that both ephrinB3(KO) and ephrinB3(lacZ) mice show an increased number of excitatory synapses, demonstrating a cytoplasmic-dependent reverse signaling role of ephrinB3 in regulating synapse number. Together, these data suggest that ephrinB3 may act like a receptor to transduce reverse signals to regulate the number of synapses formed in the hippocampus, and that it likely acts to stimulate forward signaling to modulate a number of other proteins involved in synaptic activity and learning/memory.

Eph receptor deficiencies lead to altered cochlear function.

Ephrins and Eph receptors are a family of molecules that have been implicated in many developmental processes including neuronal network formation, guidance of cell migration, and axonal pathfinding. These molecules exhibit the ability to send bidirectional signals following ligand-receptor interactions resulting from cell-cell contacts. Gene-targeted knockout mice of B-class ephrins and Eph receptors have been shown to display phenotypic responses that correlate with anatomical defects. For example, disruption of the EphB2 receptor leads to defects of the vestibular system, including pathfinding abnormalities in efferent axons and reduced endolymph production. Such developmental distortions lead to deficiencies in ionic homeostasis and repetitive circling behaviors. The present study demonstrates that B-class ephrins and Eph receptors are expressed in cochlear tissues, suggesting that they may play some role in auditory function. To determine whether ephrins and Eph receptors have a functional role in the peripheral auditory system, distortion-product otoacoustic emission (DPOAE) levels, collected across a broad frequency range, were compared between groups of mice expressing different Eph receptor genotypes. In particular, EphB1 and EphB3 receptor knockout mice exhibited significantly diminished DPOAE levels as compared to wild-type littermates, indicating that these specific Eph receptors are necessary for normal cochlear function.

Paxillin binds schwannomin and regulates its density-dependent localization and effect on cell morphology.

Neurofibromatosis type 2 is an autosomal dominant disorder characterized by tumors, predominantly schwannomas, in the nervous system. It is caused by mutations in the gene NF2, encoding the growth regulator schwannomin (also known as merlin). Mutations occur throughout the 17-exon gene, with most resulting in protein truncation and undetectable amounts of schwannomin protein. Pathogenic mutations that result in production of defective schwannomin include in-frame deletions of exon 2 and three independent missense mutations within this same exon. Mice with conditional deletion of exon 2 in Schwann cells develop schwannomas, which confirms the crucial nature of exon 2 for growth control. Here we report that the molecular adaptor paxillin binds directly to schwannomin at residues 50-70, which are encoded by exon 2. This interaction mediates the membrane localization of schwannomin to the plasma membrane, where it associates with beta 1 integrin and erbB2. It defines a pathogenic mechanism for the development of NF2 in humans with mutations in exon 2 of NF2.